Oligopeptide-20 is a growth-factor mimicking peptide used in cosmeceuticals. This article describes the development and validation of an ion-pair reversed-phase liquid chromatography method that allows, after liquid–liquid extraction, the quantification of oligopeptide-20 in cosmetic creams. Chromatographic separation was achieved on a cyanopropyl Hypersil analytical column (100 mm × 2.1 mm i.d., 5 μm particle size), using a mobile phase of acetonitrile–heptafluorobutyric acid (pH = 2.5, 9.0 mM) (70:30, v/v) containing 0.045% diethylamine at a flow rate of 0.50 mL min−1. Ultraviolet (UV) spectrophotometric detection at 225 nm was used. The method had linear calibration curve over the range 1.35–4.95 μg mL−1 for oligopeptide-20. The intra- and inter-day RSD values were less than 3.3%, while the relative percentage error, %Er, was less than 1.9. The developed method was applied successfully to the quality control of a cosmetic cream containing 0.003% (w/w) oligopeptide-20.